Enzymatic removal of burnt-on protein residues from solid surface: A potential food equipment cleanser

Burnt-on protein residues on the surfaces of the utensils used in dairy and meat processing industries possess a major challenge, where routine cleaning operations involve the use of hot alkali and/or acid, detergent washes followed by heat or chemical sanitization. These conventional cleaning approaches are neither economical nor eco-friendly. An economic enzymatic approach using alkaline protease isolated from Bacillus pseudofirmus strain SVB1 is envisaged. The efficiency of the alkaline protease (crude extract and partially purified enzyme) in removing burnt-on protein residues from solid surface was monitored by three-dimensional atomic force microscopy and protein quantification methods. In addition, the efficiency was also compared with that of the commercially available alkaline proteases. Crude alkaline protease obtained from SVB1 removed casein, dairy effluent, abattoir effluent and poultry effluents from the model solid surface efficiently. Results showed that the crude enzyme significantly removed casein when compared to the commercially available alkaline proteases. After this enzymatic cleaning and subsequent treatment, no residual enzyme activity was detected.     

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50–70 kDa), F2 (Mr:40–60 kDa) and F3 (Mr:10–15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.     

Homology Modeling Study of Bovine μ-Calpain Inhibitor-Binding Domains

The activated mammalian CAPN-structures, the CAPN/CAST complex in particular, have become an invaluable target model using the structure-based virtual screening of drug candidates from the discovery phase to development for over-activated CAPN linked to several diseases, such as post-ischemic injury and cataract formation. The effect of Ca²⁺-binding to the enzyme is thought to include activation, as well as the dissociation, aggregation, and autolysis of small regular subunits. Unfortunately, the Ca²⁺-activated enzyme tends to aggregate when provided as a divalent ion at the high-concentration required for the protease crystallization. This is also makes it very difficult to crystallize the whole-length enzyme itself, as well as the enzyme-inhibitor complex. Several parameters that influence CAPN activity have been investigated to determine its roles in Ca²⁺-modulation, autoproteolysis, phosphorylation, and intracellular distribution and inhibition by its endogenous inhibitor CAST. CAST binds and inhibits CAPN via its CAPN-inhibitor domains (four repeating domains 1–4; CAST1–4) when CAPN is activated by Ca²⁺-binding. An important key to understanding CAPN1 inhibition by CAST is to determine how CAST interacts at the molecular level with CAPN1 to inhibit its protease activity. In this study, a 3D structure model of a CAPN1 bound bovine CAST4 complex was built by comparative modeling based on the only known template structure of a rat CAPN2/CAST4 complex. The complex model suggests certain residues of bovine CAST4, notably, the TIPPKYQ motif sequence, and the structural elements of these residues, which are important for CAPN1 inhibition. In particular, as CAST4 docks near the flexible active site of CAPN1, conformational changes at the interaction site after binding could be directly related to CAST4 inhibitory activity. These functional interfaces can serve as a guide to the site-mutagenesis in research on bovine CAPN1 structure-function relationships for the design of small molecules inhibitors to prevent uncontrolled and unspecific degradation in the proteolysis of key protease substrates.     

Sugarcane Serine Peptidase Inhibitors,Serine Peptidases,and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding

Sugarcane’s (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.     

碱性蛋白酶洗涤稳定性提高的研究进展

碱性蛋白酶是液体洗涤剂中使用量最大的一类酶制剂,应用于洗涤剂行业的碱性蛋白酶占碱性蛋白酶市场的60%以上。不同于粉状洗涤剂中的酶制剂被造粒所包裹,液体洗涤剂中的碱性蛋白酶直接暴露于溶液中,与洗涤剂成分如表面活性剂、洗涤助剂及漂白剂直接接触作用,使得蛋白酶洗涤稳定性下降。除此之外,蛋白酶在液体洗涤剂环境中存在普遍的自溶失活现象,对其洗涤性能造成不利影响。如何提升碱性蛋白酶的稳定性是液体洗涤剂领域中的一个热点问题。本文介绍了碱性蛋白酶的催化与失活机制,综述了几种常用于稳定碱性蛋白酶的策略,即添加稳定剂、分子改造和化学修饰,重点介绍了化学修饰中的聚乙二醇化修饰与多糖修饰,对比了两种修饰方法的过程与效果,并在最后对碱性蛋白酶稳定性提高策略进行了展望。     

A Taguchi design approach for the enhancement of a detergent-biocompatible alkaline thermostable protease production by Streptomyces mutabilis strain TN-X30

The ability of microorganisms to grow at high temperature, alkaline pH, and high salinity makes them an attractive target for enzyme-production with several industrial applications. One strain TN-X30 has been selected as protease producer and identified as Streptomyces mutabilis after a phenotypic and molecular study. Its production of protease was improved using Taguchi L27 design. The strategy was carried out to identify the optimum levels and the interaction of the screened factors. Following this step, maximum protease activity (10,895 U/ml) was achieved after 6-days of incubation. The TN-X30 protease activity had an optimum of pH and temperature of 10 and 65°C, respectively. Thermodynamic parameters at 60°C were enthalpy 14.26 kJ/mol, entropy −220 J/mol/K, and Gibbs free energy 90.53 kJ/mol. TN-X30 protease production displayed a 16-fold increase reaching 175,000 U/ml in a 100-L fermentor. Furthermore, the lyophilization in presence of sorbitol enhanced the stability of the TN-X30 protease which remained active at 75% after 24-months of storage. The lyophilized TN-X30 protease exhibited exceptional stability indexes in presence of some known commercialized detergent components as NEODOL® 25-7, Dehydol® LT 7, Na₂ CMC, Galaxy LAS, Galaxy LES 70, Galaxy 110, Galaxy CAPB Plus, and Sulfacid K. The lyophilized enzyme also displayed high stability with respect to both solid and liquid detergents. Finally, TN-X30 protease exhibited remarkable destaining of blood, egg, and chocolate stained cloth pieces. These findings may promote TN-X30 protease for use as bioadditive in detergent formulation, thereby reducing environmental chemical threat.     

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro) and the papain-like protease (PL^pro), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro. Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro, highlighting the potential for dual PL^pro/M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.     

猪血蛋白水解液氨基酸成分分析

以新鲜猪血为原料,制得了猪血蛋白干粉,选用三种不同类别的蛋白酶在适宜的条件下水解猪血蛋白干粉,并得到精制水解蛋白液。用氨基酸分析仪检测水解液中氨基酸种类及含量,并与医用脑蛋白液进行比较分析。结果表明胰蛋白酶水解效果最好,水解液成分与医用脑蛋白液成分比较接近。